Regioselective hydroxylation of isophorone

ABSTRACT

The present invention relates to novel process for the production of ketoisophorone via biocatalytic conversion of isophorone, in particular a one-pot biocatalytic system for conversion of α-isophorone in a two-step oxidation process, with a first oxidation being catalyzed by a heme containing oxidoreductase such as a cytochrome P450 monooxygenase followed by another oxidation which can be either a chemical reaction or a biocatalytic reaction, in particular wherein the oxidation is catalyzed by an NAD(P) or NADP(H)-dependent oxidoreductase. The invention further provides polypeptides and nucleic acid sequences coding for cytochrome P450 monooxygenase with modified (higher) substrate selectivity, total turnover numbers and/or (re)activity compared to the wild-type enzyme. Ketoisophorone is useful as building block in the synthesis of vitamins and carotenoids.

The present invention relates to novel process for the production ofketoisophorone via biocatalytic conversion of isophorone, in particulara one-pot biocatalytic system for conversion of α-isophorone in atwo-step oxidation process, with a first oxidation being catalyzed by aheme containing oxidoreductase such as a cytochrome P450 monooxygenasefollowed by another oxidation which can be either a chemical reaction ora biocatalytic reaction, in particular wherein the oxidation iscatalyzed by an NAD(P) or NADP(H)-dependent oxidoreductase. Theinvention further provides polypeptides and nucleic acid sequencescoding for cytochrome P450 monooxygenase with modified (higher)substrate selectivity, total turnover numbers and/or (re)activitycompared to the wild-type enzyme. Ketoisophorone is useful as buildingblock in the synthesis of vitamins and carotenoids.

Monooxygenated terpenoids such as e.g. isophorone are relevant targetcompounds for the synthesis of active pharmaceutical ingredients (APIs),fragrances and nutritional supplements, e.g. vitamins. An example ofsuch a target compound is ketoisophorone(2,6,6-trimethylcyclohex-2-ene-1,4-dione; KIP) which can be synthesizedby oxidation of isophorone (IP). KIP can be further isomerized totrimethyhydroquinone (TMHQ), the key building block in the synthesis ofvitamin E (for more detail see e.g. Ullmann's Encyclopedia of IndustrialChemistry, 6^(th), completely revised edition, Volume 38, Wiley-VCH,2002). Additionally, KIP also may serve as important precursor ofseveral carotenoids such as e.g. via the catalytic action of levodionereductase, whereby KIP is converted into the chiral intermediate(4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (actinol).

Unfortunately, the generation of TMHQ as known today is a time, materialand cost intensive process which requires high temperatures and/ororganic solvents, toxic heavy metal catalysts, with generation ofundesired by-products.

Although the starting material α-IP is readily available, the knownchemical conversion into KIP is a very ineffective process, with firstisomerization of α-IP into ß-IP followed by homogeneous liquid oxidationto KIP, a process with the equilibrium heavily shifted towards α-IP: nomore than 2% of α-IP are converted into ß-IP. Until now, there is noalternative, e.g. biocatalytic reaction, known which would lead to highyields of KIP via regioselective oxidation of α-IP.

Thus, there is a strong need to improve the known vitamin E synthesis,in particular to improve the production of KIP as one of thekey-building blocks from its precursor, e.g. to reduce reaction andpurification steps and/or to reduce waste and process energy demands,but in particular to improve the regioselectivity and total turnovernumbers (TTN) of said reactions, especially in the oxidative conversionof α-IP to KIP.

Surprisingly, we now found a new biocatalytic route to KIP, i.e. acascade system wherein KIP is generated from α-IP via a double allyticoxidation (FIG. 1A). Preferably, the first step is an enzymaticconversion using a heme containing oxidoreductase enzyme havingcytochrome P450 enzyme activity followed by a second step, which mightbe also an enzymatic conversion, in particular via NADP(H) orNAD(H)-dependent enzymes, such as e.g. NADP(H) or NAD(H)-dependentoxidoreductases, or a chemical oxidation step as known to the personskilled in the art.

In particular, the present invention provides a two-step oxidationprocess, wherein in step 1 α-IP is converted to 4-hydroxy-α-isophorone(HIP) via biocatalytic action of a cytochrome P450 enzyme and wherein instep 2 said HIP is converted, i.e. oxidized, into KIP, said step 2 beingeither a chemical reaction or being catalyzed via action of a suitableenzyme. Preferably, both steps are performed as one-pot multi-enzymatictransformation reaction. With this approach, we were able to obtainconversion rates in the range of at least 60%, such as e.g. 70%, 80%,90%, 95% or higher, i.e. up to 100% conversion.

Cytochrome P450 enzymes (CYPs or P450s) are a diverse superfamily ofheme oxidoreductases capable of performing many oxidative reactions,most notably the insertion of oxygen into a chemically inert C-H bond,using oxygen as a benign oxidant and releasing water as by-product.During this catalytic cycle, oxygen activation is enabled by twoelectrons supplied by NAD(P)H and shuttled by redox partners. Directaromatic ring hydroxylation is a synthetically attractive reaction whichhas been reported for several P450 enzymes. A well-known example of suchP450 enzyme is the cytochrome P450 CYP102A1 from Bacillus megateriumcommonly referred to as the P450 BM3. P450 BM3 is a water-soluble enzymeof 118 kDa. Until now, these enzymes have been hardly used inbiotechnical processes, since they are difficult to express in theestablished host systems and are rather sensitive to inactivation in theisolated state.

The term “TTN”, which is art-recognized, is defined as the ratio ofproduct concentration and enzyme concentration calculated over 24 h, andis a determining feature of enzyme activity. TTN can be measured byHPLC, in particular through calibration curves (product formed) and COdifference spectroscopy (enzyme concentration). The method is known inthe art. The improvement of the TTN values is one particular subject ofthe present application.

In one embodiment, the present invention provides a biological processfor the conversion of α-IP to 4-hydroxy-α-isophorone (HIP), saidconversion being catalyzed by an enzyme having P450 monooxygenaseactivity. Thus, α-IP is incubated in the presence of said enzyme undersuitable conditions as defined herein for step 1.

Preferably, said biocatalytic conversion is the first part of a doubleoxidation cascade, wherein in step 1 α-IP is biocatalytic oxidized to(HIP), which is subsequently oxidized in step 2 (as defined below) intoKIP.

As used herein, the terms “enzyme”, “P450 monooxygenase”, “cytochromeP450 monooxygenase” or “P450 enzyme” are used interchangeably herein inconnection with the description of the present invention.

The enzyme used for step 1 may include any P450 monooxygenase includingenzymes isolated from microorganisms, yeast or mammals. Preferably, thebiocatalyst/enzyme is selected from the P450 monooxygenase of Bacillus,Geobacillus, Pseudomonas, Erythrobacter, Burkholderia, Herpetosiphon,Ralstonia, Bradyrhizobium, Azorhizobium, Streptomyces, Rhodopseudomonas,Rhodococcus, Delftia, Saccharopolyspora, Comamonas, Burkholderia,Cupriavidus, Variovorax, Fusarium, Gibberella, Aspergillus orAmycolatopsis, more preferably selected from Bacillus megaterium,Bacillus subtilis, Bacillus licheniformis, Bacillus weihenstephanensis,Bacillus cereus, Bacillus anthracis, Pseudomonas putida, Burkholderiasp. 383, Erythrobacter litoralis, Geobacillus sp. Y412MC10,Herpetosiphon aurantiacus, Ralstonia eutropha, Ralstonia pickettii,Ralstonia metallidurans, Bradyrhizobium japonicum, Azorhizobiumcaulinodans, Streptomyces avermitilis, Rhodopseudomonas palustris,Rhodococcus sp., Rhodococcus ruber, Rhodococcus sp. NCIMB 9784, Delftiaacidovorans, Saccharopolyspora erythraea, Comamonas testosteroni,Burkholderia mallei, Cupriavidus taiwanensis, Variovorax paradoxus,Fusarium oxysporum, Gibberella zeae, Gibberella moniliformis,Aspergillus fumigatus or Amycolatopsis orientalis, most preferablyBacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillusweihenstephanensis, Bacillus cereus, Bacillus anthracis, Pseudomonasputida, Rhodococcus sp., in particular Bacillus megaterium or Bacillussubtilis, such as Bacillus megaterium (P450 BM3, CYP102A1) shown in SEQID NO:1 or homologous sequences thereof showing the same enzymaticactivity. Thus, the present invention is directed to a process asdescribed herein wherein an enzyme having at least 35%, such as 40, 50,60, 70, 75, 80, 85, 90, 95, 98, 99% identity to SEQ ID NO:1, theidentity being determined over the entire amino acid sequence usingClustalW2 in the default settings of 24 Nov. 2009. An example of auseful mammalian enzyme is the human CYP2D6. Furthermore, preferred arechimeric enzymes of two or more P450 enzymes or at least domains of suchenzymes, such as e.g. the heme domain from one enzyme and/or theNADPH-dependent reductase domain from another enzyme. A particularuseful chimeric P450 enzyme is the self-sufficient P450cam-RhFRed P450as shown in SEQ ID NO:3, encoded by a nucleic sequence including SEQIDNO:4. Thus, the present invention is directed to a process as describedherein wherein an enzyme having at least 62%, such as 70, 75, 80, 85,90, 95, 98, 99% identity to SEQ ID NO:3, the identity being determinedover the entire amino acid sequence of the reductase domain part RhFRedusing ClustalW2 in the default settings of 24 Nov. 2009.

In one particular embodiment, step 1 according to the present inventionis performed using the P450 BM3 (EC 1.6.2.4) as shown in SEQ ID NO:1,encoded e.g. by the nucleic acid sequence shown in SEQID NO:2.

In one particular embodiment of step 1 as defined herein, the chimericself-sufficient P450cam-RhFRed enzyme is used consisting of the hemedomain of the well-known CYP101A1 (P450cam, camphor 5-monooxygenase)from Pseudomonas putida fused to the NADPH-dependent reductase domain(RhFRed) of CYP116B2 from Rhodococcus sp. (P450RhF), see Robin et al.,Beilstein J. Org. Chem. 2011, 7, 1494-1498 or O'Reilly et al., Catal.Sci. Technol. Catal Sci. Technol 2013, 3, 1490-1492.

A biocatalytic process of the present invention including e.g. step 1and/or step 2 might be performed using either wild-type enzymesderivable from nature or modified or mutated, i.e. optimized, enzymes,such as modified or chimeric (i.e. “synthetic”) P450 enzymes.Optimization also includes modification which enables high expression ina specific host system, which is a standard method and known in the art.

The term “wild-type” in the context of the present invention may includeboth P450 monooxygenase sequences derivable from nature as well asvariants of synthetic (chimeric) P450 enzymes. The terms “wild-typeP450” and “non-modified P450” are used interchangeably herein.

A “mutant”, “mutant enzyme”, “mutant P450 enzyme” or “P450 variant” suchas P450 BM3 or P450cam-RhFRed modified enzyme, may include any variantderivable from a given wild-type or chimeric (in the sense as definedherein) enzyme/P450 (according to the above definition) according to theteachings of the present invention and being more active/efficient ine.g. regio-selectivity and/or TTN than the respective wild-type enzyme.For the scope of the present invention, it is not relevant how themutant(s) is/are obtained; such mutants may be obtained, e.g., bysite-directed mutagenesis, saturation mutagenesis, randommutagenesis/directed evolution, chemical or UV mutagenesis of entirecells/organisms, and other methods which are known in the art. Thesemutants may also be generated, e.g., by designing synthetic genes,and/or produced by in vitro (cell-free) translation. For testing ofspecific activity, mutants may be (over-)expressed by methods known tothose skilled in the art. Different test assays are available, such ase.g. pNCA-assays, 4-AAP-assay or pNTP-assay which are all known to theskilled person. The terms “mutant P450 enzyme” and “modified P450enzyme” are used interchangeably herein.

Thus, in one aspect the present invention is directed to a modifiedenzyme having P450 activity, in particular a P450 enzyme,showing—compared to the wild-type or non-modified enzyme—improvedregio-enantioselectivity, improved enantiomeric excess values (ee)and/or improved TTN. Such mutation or modification is said to be afunctional mutation.

Depending on the used enzyme in step 1, α-IP might be converted mainlyinto (R)-HIP, with e.g. an ee towards (R)-HIP (as compared to HIP) of atleast 40%, such as at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or even at least 99%. Step 1 as performed with a P450 mutant as definedherein might lead to an ee towards (R)-HIP of up to 99%.

The mutant P450 as according to the present invention are improved inthe TTN, with preferred TTN in the range of at least 1 to 1.5 g/l/h,such as e.g. at least 2 g/l/h leading to a concentration of the reactionproduct in the range of 50 g/l after 24 h. More preferred are TTN valuesin the range of at least 10000 to 13000, such as e.g. at least 50000 to100000. With regards to the improvement of TTN, the mutant P450 enzymesaccording to the present invention show an increase of at least 2-fold,such as at least 3, 4, 5, in particular at least 6-fold increase in TTNcompared to the respective wild-type or non-modified P450 enzymes. Thismight be event increased by at least 10-fold.

The P450 enzyme used to generate the modified version according to oneaspect of the present invention and which can be preferably used in thestep 1 oxidation might be any P450 enzyme including chimeric forms, aslong as the introduction of one or more mutations results in animprovement of regio-enantioselectivity, ee and/or TTN, i.e. improvedbioconversion of α-IP into HIP-one intermediate generated in step 1 ofthe inventive process (see FIG. 1B). In particular, the modified P450enzyme might be based on the wild-type P450 BM3 or P450cam-RhFRed,preferably the P450cam-RhFRed.

In a particular embodiment, the modified P450 enzyme produces at least10 mmol/l corresponding to 1.5 g/l of HIP generated by biocatalyticoxygenation of α-IP. With regards to efficiency of conversion, step 1 ofthe bioconversion of α-IP to HIP using enzymes as described herein mightbe carried out with a conversion rate of at least about 60%, preferablyat least about 70%, 75%, 80%, 90%, 95%, 98% conversion. As preferablystep 2 is performed with the same conversion rates, more preferably acomplete conversion from HIP to KIP, said conversion rates might applyto the complete double oxidation process, i.e. the cascade process fromα-IP to KIP.

In one preferred embodiment, the at least one mutation is at one or moreamino acid positions selected from the group consisting of amino acidpositions corresponding to positions 87, 244, 247 and combinationsthereof of the amino acid sequence of chimeric self-sufficientP450cam-RhFRed as shown in SEQID NO:3, defined as the wild-type sequence(this sequence already carries the amino acid substitution Y96F). Morepreferably, the wild-type P450cam-RhFRed is mutated wherein the mutatedor modified enzyme comprises a mutation in one or more of the amino acidresidues F87, L244 and/or V247 (in addition to Y96F which is alreadypresent in the parent or wild-type chimeric enzyme). Particularlypreferred are amino acid substitutions but also other forms of mutationsknown to the skilled person are possible as long as they result inenzymes having improved properties as defined herein.

Preferably, the modified P450 enzyme such as the modified P450cam-RhFRedcomprises amino acid substitutions on at least one position selectedfrom F87, L244 and/or V247 shown in the wild-type chimeric enzyme (whichalready carries substitution Y96F).

With regards to preferred amino acid residues on position 244, thepresence of a polar amino acid residue, e.g. asparagine, serine orcysteine or positively charged residues, e.g. histidine, leads to animprovement in ee and/or TTN values compared to L244 and according tothe present invention. Preferably, the amino acid on positioncorresponding to position L244 in the wild-type chimeric P450cam-RhFRedshown in SEQ ID NO:3 is selected from alanine, asparagine, serine,glycine, isoleucine, cysteine, tyrosine or histidine, with the mostpreferred amino acid residue being adenine on position 244, e.g.substitution L244A.

With regards to preferred amino acid residues on position 247, thepresence of an apolar side chain or the polar amino acid residue such ase.g. asparagine or serine leads to an improvement in ee and/or TTNvalues compared to V247 and according to the present invention.Preferably, the amino acid on position corresponding to position V247 inthe wild-type chimeric P450cam-RhFRed shown in SEQ ID NO:3 is selectedfrom lysine, phenylalanine, isoleucine or serine, with the mostpreferred amino acid residue being leucine on position 247, e.g.substitution being V247L.

With regards to preferred amino acid residues on position 87, there seemto be no preference for a specific amino acid leading to an improvementin step 1 of the inventive process. A preferred amino acid residue onposition 87 is tryptophan, e.g. substitution F87W.

In one aspect, the mutated enzyme which might be used in step 1 is amodified P450 enzyme such as e.g. a modified P450cam-RhFRed comprising acombination of at least 2 mutations mentioned above, such as e.g. aminoacid substitutions on positions corresponding to positions 244 and 247in the wild-type chimeric P450cam-RhFRed according to SEQ ID NO:3, suchas e.g. L244-V247 shown in SEQ ID NO:3, preferably a combination ofsubstitutions is L244S-V247L, L244N-V247L, L244G-V247L, L244I-V247L,L244C-V247L, L244I-V247F, L244Y-V247F, L244N-V247F, L244H-V247F,L244G-V247F, L244I-V247I, L244I-V247S, or L244A-V247L, with mostpreferred being the combination L244A-V247L.

According to another aspect of the present invention, the mutated enzymewhich might be used in step 1 is a modified P450 enzyme such as e.g.modified P450cam-RhFRed comprising a combination of at least 3 mutationsmentioned above, such as e.g. amino acid substitutions on positions 244,247 and 87, such as e.g. F87-L244-V247 shown in SEQ ID NO:3, preferablya combination of substitutions F87W-L244A-V247L, but also includes anyof the above mentioned double amino acid substitutions on positions 244and 247 in combination with preferably F87W.

The modified P450 enzyme according to step 1 of the present invention,such as e.g. modified chimeric P450cam-RhFRed, preferably comprises atleast 1, at least 2 or at least 3 mutations, e.g. substitutions, on oneof the above-identified positions when compared with the amino acidsequence of the corresponding non-modified chimeric enzyme asexemplified by SEQ ID NO:3. In case of amino acid substitutions on atleast 3 of the herein-identified residues, preferably the combination ofamino acid substitutions F87W-L244A-V247L, leading to an ee of 99%towards (R)-HIP from α-IP.

All these modifications mentioned above leading to a mutant P450 enzyme,in particular a mutant P450cam-RhFRed, might be used in a process forconversion, i.e. oxidation of α-IP into HIP, in particular for step 1 ofthe biocatalytic double oxidation, preferably one-pot biocatalyticdouble oxidation, as described herein.

The present invention features in one aspect nucleic acid sequencescoding for the novel modified P450 enzymes as being part of the presentinvention as well as to vectors or systems used to express such modifiedenzymes in a suitable host system. The skilled person knows suchvectors, hosts and the corresponding systems for expression of enzymes.

As used herein, the present invention also encompasses P450 enzymes withat least one of the abovementioned sequence positions, but additionallywhich carry amino acid substitution(s) other than the one mentionedspecifically, but still lead to a mutant which, like the mutant whichhas been mentioned specifically, show the same properties with respectto the wild-type enzyme and catalyze at least one of the abovementionedhydroxylation reactions. Such mutations are also called “silentmutations”, which do not alter the activity of the mutants as describedherein.

The process as of step 1 cascade process as defined herein using mutantP450 enzymes according to the present invention, in particular therecombinant P450cam-RhFRed mutants, results in increased specificactivity in the oxidation of α-IP resulting in HIP with improved TTNvalues and (enantio)selectivity for production of HIP from α-IP.

One particular aspect of the present invention is a two-step oxidationprocess, wherein step 1 as described above consisting of the conversionof α-IP to HIP using a heme containing oxidoreductase such as a P450enzyme is followed by a subsequent oxidation, i.e. step 2 of the cascadeoxidation as described herein. Step 2 might be a chemical oxidationknown to the skilled person or, preferably, a further biocatalyticoxidation using a suitable enzyme for conversion of HIP into KIP (seeFIGS. 1A and 1C).

In a preferred embodiment, step 2 according to the present invention isa biocatalytic conversion of HIP into KIP via the activity of a suitableenzyme, such as e.g. NADP(H) or NAD(P) dependent enzymes, includingNADP(H) or NAD(P) dependent oxidoreductases (EC 1), preferably enzymeshaving activity as alcohol dehydrogenase, carbonyl reductase, ketoreductase or monooxygenase, in particular alcohol dehydrogenase (ADH) orcarbonyl reductase (CR) activity. More preferably, the enzyme isselected from ADH of Candida magnoliae, such as e.g. Cm-ADH10 (GenBankaccession no. AGA42262.1), or carbonyl reductase from Sporobolomycessalmonicolor (UniProt accession no. Q9UUN9).

As used herein, the biocatalytic processes as of step 1 and/or step 2can be carried out with either wild-type enzymes derivable from natureor modified or mutated, i.e. optimized, enzymes. Step 1 and/or step 2might be performed with isolated/purified enzymes or as a whole cellbiocatalyst in a biotransformation reaction, wherein the enzyme(s)is/are expressed in a suitable host system as described herein.

With regards to the two-step process as defined herein, in oneembodiment the biotransformation of step 1 followed by step 2 arecarried out separately, i.e. not as a one-pot biocatalytic system. Inthis case, either step 1 and step 2 are running in the presence ofsuitable co-substrates added to the reaction, such as e.g. glucose,isopropanol or phosphite as suitable co-substrates for step 1, and/ore.g. acetone, chloroacetone, ethyl acetoacetate, ethyl levulinate,chloroacetone or ethyl acetoacetate, preferably chloroacetone or ethylacetoacetate as suitable co-substrates for step 2. These co-substratesare combined with the enzymes used for step 1 and/or step 2 as definedherein.

Preferably, the co-substrates are present in an amount of at least 1equivalent, more preferably in an amount of between 1 and 2 equivalentscompared to the substrate, such as α-IP or HIP in the case of step 1 andstep 2, respectively.

In one embodiment, the bioconversion or biotransformation orbiocatalytic reaction (as interchangeably used herein) according to step2 is carried out in the presence of an alcohol dehydrogenase, inparticular ADH from Candida magnoliae, preferably Cm-ADH10 according toSEQID NO:5, which can be expressed from a nucleic acid sequenceaccording to SEQ ID NO:6. Thus, HIP is incubated in the presence of saidenzyme under suitable conditions as defined herein for step 2.

In a further embodiment, the bioconversion according to step 2 iscarried out in the presence of a carbonyl reductase from Sporobolomycessalmonicolor (55CR), in particular a carbonyl reductase according toSEQID NO:7, which can be expressed from a nucleic acid sequenceaccording to SEQ ID NO:8.

The preferred enzyme to be used for step 2 is an ADH, more preferablyCm-ADH10, particularly in concentrations of about 0.5 to 1.5 mg/ml,preferably of about 1 mg/ml when used as purified enzyme.

In one aspect of the present invention there is provided a two-stepoxidation process, wherein both steps are preferably catalyzed bysuitable enzymes as described herein. Both enzymes or only one of theenzymes might be used in isolated, i.e. purified form. In a preferreddouble oxidation process according to the present invention, the processis performed as a biotransformation, wherein both enzymes to be used instep 1 and step 2 are present/expressed in a suitable host system. Thisdouble oxidation process might be carried out separately, i.e. theenzymes being expressed independently, or, which is preferred, theenzymes to be used in step 1 and step 2 are expressed in the same hostand used as a one-pot reaction.

Expression of the enzymes used according to the present invention can beachieved in any host system, including (micro)organisms, which allowsexpression of the nucleic acids according to the invention, includingfunctional equivalents or derivatives or mutants. Examples of suitablehost (micro)organisms are bacteria, fungi, yeasts or plant or animalcells. Preferred organisms are bacteria such as those of the generaEscherichia, such as, for example, Escherichia coli, Streptomyces,Bacillus, Rhodococcus, such as for example Rhodococcus erythropolis,Rhodococcus rhodochrous, Rhodococcus ruber, Rhodococcus equi, orPseudomonas, such as for example Pseudomonas putida, or eukaryoticmicroorganisms such as Saccharomyces, such as Saccharomyces cerevisiae,Aspergillus, such as Aspergillus niger, Pichia, such as Pichia pastoris,Hansenula, such as Hansenula polymorpha or Yarrowia, such as Yarrowialipolytica and higher eukaryotic cells from animals or plants. Inparticularly preferred is Escherichia, Bacillus, Pseudomonas orRhodococcus, more particularly preferred is Escherichia coli B, such asE. coli BL21 (DE3) or other derivatives, or E. coli K-12 strains.

Independently of the expression in a one pot or two pot system, thebiotransformation of step 1 and/or step 2 as defined herein isparticularly performed at a temperature in the range of from 10° C. to50° C., preferably between 20° C. and 40° C., such as e.g. about 30° C.to 40° C.

As used herein, and independently of the expression in a one pot or twopot system, the biotransformation of step 1 and/or step 2 as definedherein is in particular performed at a pH of from 4.0 to 10.0,preferably of from 5.7 to 8.0, in particular of from 7.0 to 8.0.

As used herein, and independently of the expression in a one pot or twopot system, the biotransformation of step 1 and/or step 2 as definedherein is particularly performed in a buffer selected from phosphate,such as potassium carbonate, acetate or Tris-HCl. The skilled personwill know which buffer is most suitable with regards to the usedenzymes.

As used herein, a process according to the present invention—and inparticular step 1—is performed in the presence of preferably 2 to 6 μMP450 enzyme, either the wild-type enzyme or the mutants as definedherein. With regards to step 2 of the present invention, theconcentration of enzyme, such as e.g. ADH, preferably Cm-ADH10, theconcentration may vary. Optionally, the one pot reaction might becarried out in the presence of 10 mg/ml glucose as co-substrate withoptional addition of co-factors such as NADP(H) or NAD(H).

Typically, a process according to the present invention including step 1and step 2 and independently of the expression in a one pot or two potsystem, runs between 1 h up to 48 h, preferably in the range of from 18h to 24 h.

Thus according to one embodiment, the present invention relates to abiocatalytic process for the conversion of α-isophorone to4-hydroxy-α-isophorone (HIP), wherein α-isophorone is incubated undersuitable conditions in the presence of a heme containing oxidoreductase,preferably an enzyme having P450 monooxygenase activity, more preferablyselected from P450cam-RhFRed or Bacillus megaterium, even morepreferably a polypeptide according to a sequence having at least 35%identity to SEQ ID NO:1 or a sequence with at least 62% identity to SEQID NO:3, said suitable conditions include incubation at pH of from 4.0to 10.0 for 1 to 48 h with optionally isolation of HIP from the reactionmixture.

Thus, according to a further embodiment, the present invention relatesto a biocatalytic process for the production of ketoisophorone (KIP),wherein the product from step 2 is incubated under suitable conditionsin the in the presence of a NAD(H) or NADP(H)-dependent enzyme,preferably NAD(H) or NADP(H)-dependent oxidoreductase (EC 1), morepreferably an enzyme having activity as alcohol dehydrogenase, carbonylreductase, keto reductase or monooxygenase, even more preferably havingalcohol dehydrogenase or carbonyl reductase activity, most preferably analcohol dehydrogenase from Candida magnoliae or a carbonyl reductasefrom Sporobolomyces salmonicolor, said suitable conditions includeincubation at pH of from 4.0 to 10.0 for 1 to 48 h with optionallyisolation of KIP from the reaction mixture.

As used herein, the terms “one-pot” means that the process as describedherein including step 1 and step 2 is carried out in the same reactor(i.e. the same reaction buffer under the same conditions), i.e. there isonly one reaction comprising 2 steps. The “same reactor” is defined asonly the same reaction “pot” and/or the same host organism. It mightfurthermore mean that the polynucleotides encoding both enzymes to beused in step 1 and step 2 are cloned on the same vector or are bothintegrated in the genome of the (same) host organism. In this case, theprocess is preferably carried out as simultaneous single-step process asdefined below.

The term “double oxidation”, “double allylic oxidation”, “cascadeoxidation”, “double oxidation cascade” are used herein interchangeableand describe two oxidation steps, leading to conversion of α-IP to HIP,i.e. step 1 as defined herein, and further to KIP, i.e. step 2 asdefined herein (see FIG. 1A). These 2 oxidations might be performedsimultaneously, i.e. might occur at the same time, which would bedefined also defined as “one-step process”, or might be subsequently,i.e. step 1 is followed by step 2 (either in a one-pot or two-pot systemas defined above), which would be defined as “two-step process”, whichis the preferred method.

The term “HIP” as used herein includes both enantiomers in any possibleratio, i.e. mixtures of either (R)- and (S)-HIP as well as the singleenantiomers, without any enantiomeric preference with regards to thepresent invention. Depending on the P450 enzyme, an ee towards either(R)- or (S)-HIP might be generated. For example, use of the mutant P450in step 1 as described herein leads to an ee of up to 99% towards(R)-HIP. In case step 1 would be performed in the presence of P450 BM3this preference towards (R)-HIP would be less prominent, such as e.g. inthe range of about 80% or less. The HIP obtained via step 1 is furtherconverted into KIP in the presence of an enzyme as defined herein,wherein said enzyme is capable of using HIP as defined herein assubstrate (and optionally further co-substrates or co-factors),including a substrate which is selected from (R)-HIP, (S)-HIP, or amixture of (R)/(S)-HIP, including mixtures with an ee in the range of50, 60, 70, 80, 90, 95, 98, 99% towards (R)-HIP.

As used herein, the term “specific activity” or “activity” with regardsto enzymes means its catalytic activity, i.e. its ability to catalyzeformation of a product from a given substrate. The specific activitydefines the amount of substrate consumed and/or product produced in agiven time period and per defined amount of protein at a definedtemperature. Typically, specific activity is expressed in μmol substrateconsumed or product formed per min per mg of protein. Typically,μmol/min is abbreviated by U (=unit). Therefore, the unit definitionsfor specific activity of μmol/min/(mg of protein) or U/(mg of protein)are used interchangeably throughout this document.

The term “vitamin E” is used herein as a generic descriptor for alltocopherol and tocotrienol derivatives exhibiting qualitatively thebiological activity of α-tocopherol (IUPAC-IUB Recommendation 1981, Eur.J. Biochem. 123, 473-475, 1982).

In connection with the present invention it is understood that thementioned microorganisms also include synonyms or basonyms of suchspecies having the same physiological properties, as defined by theInternational Code of Nomenclature of Prokaryotes. The nomenclature ofthe microorganisms as used herein is the one officially accepted (at thefiling date of the priority application) by the International Committeeon Systematics of Prokaryotes and the Bacteriology and AppliedMicrobiology Division of the International Union of MicrobiologicalSocieties, and published by its official publication vehicleInternational Journal of Systematic and Evolutionary Microbiology(IJSEM). This applies e.g. to Sporobolomyces salmonicolor, which is alsoknown under the synonym (and as used interchangeably herein)Sporidiobolus salmonicolor.

The present invention includes the following non-limiting embodiments:

(1) A biocatalytic process for the conversion of α-isophorone to4-hydroxy-α-isophorone (HIP), wherein α-isophorone is incubated undersuitable conditions in the presence of a heme containing oxidoreductase,preferably an enzyme having P450 monooxygenase activity, more preferablyselected from P450cam-RhFRed or Bacillus megaterium, even morepreferably a polypeptide according to a sequence having at least 35%identity to SEQ ID NO:1 or a sequence with at least 62% identity to SEQID NO:3, said suitable conditions include incubation at pH of from 4.0to 10.0 for 1 to 48 h with optionally isolation of HIP from the reactionmixture.

(2) A biocatalytic process for the production of ketoisophorone (KIP),wherein the product from step 2 is incubated under suitable conditionsin the in the presence of a NAD(H) or NADP(H)-dependent enzyme,preferably NAD(H) or NADP(H)-dependent oxidoreductase (EC 1), morepreferably an enzyme having activity as alcohol dehydrogenase, carbonylreductase, keto reductase or monooxygenase, even more preferably havingalcohol dehydrogenase or carbonyl reductase activity, most preferably analcohol dehydrogenase from Candida magnoliae or a carbonyl reductasefrom Sporobolomyces salmonicolor, said suitable conditions includeincubation at pH of from 4.0 to 10.0 for 1 to 48 h with optionallyisolation of KIP from the reaction mixture.

(3) A process for the conversion of α-isophorone into KIP, wherein theprocess of embodiment (1) is followed by the process of embodiment (2).

(4) A process according to any of the embodiments (1), (2) or (3),wherein the biocatalytic process for the conversion of α-isophorone to4-hydroxy-α-isophorone (HIP) and/or the conversion of HIP toketoisophorone (KIP) is carried out in the presence of (a) aco-substrate selected from the group consisting of glucose, isopropanoland phosphite with regards to conversion of α-isophorone to HIP; or (b)a co-substrate selected from the group consisting of acetone,chloroacetone, ethyl acetoacetate, ethyl levulinate, chloroacetone andethyl acetoacetate with regards to conversion of HIP to KIP.

(5) A process for the conversion of α-isophorone into KIP, wherein theprocess of embodiment (1) is followed by a chemical conversion of HIPinto KIP.

(6) A process according to any of the embodiments (1), (2), (3) or (4),wherein the process is performed in a one-pot biocatalytic system.

(7) A process according to any of the embodiments (1), (2), (3), (4),(5) or (6), wherein the conversion rate is at least 60%.

(8) A modified P450 monooxygenase capable of regio- and stereoselectivehydroxylation of α-isophorone into HIP, wherein the amino acid sequencecomprises one or more mutation(s) on a position corresponding toposition(s) 96, 87, 244, 247, and/or combinations thereof of aP450cam-RhFRed P450 monooxygenase according to SEQ ID NO:3, and whereinthe total turnover number is increased by at least 2-fold compared tothe respective non-modified P450 monooxygenase.

(9) A modified P450 monooxygenase according to embodiment (8), wherein:

(a) the introduced amino acid on a position corresponding to position244 are selected from the group consisting of alanine, asparagine,serine, glycine, isoleucine, cysteine, tyrosine and histidine,preferably from alanine; and/or

(b) the introduced amino acid on a position corresponding to position247 are selected from the group consisting of lysine, phenylalanine,isoleucine and serine, preferably from lysine; and/or

(c) the introduced amino acid on a position corresponding to position 87is tryptophan; and/or

(d) the introduced amino acid on a position corresponding to position 97is phenylalanine.

(10) A biocatalytic process according to any of embodiments (1), (2),(3), (4) (5), (6) or (7), wherein the conversion of α-isophorone to HIPis carried out with a modified P450 monooxygenase according toembodiments (8) or (9).

(11) A polynucleotide sequence sequence comprising a DNA sequence codingfor a P450 monooxygenase as of embodiments (8) or (9).

(12) A host cell wherein a P450 monooxygenase according to embodiments(8) or (9) is expressed, preferably the host being selected from thegroup consisting of bacteria, fungi, yeasts or plant or animal cells,more preferably selected from Escherichia, Streptomyces, Bacillus,Rhodococcus, Pseudomonas, Saccharomyces, Aspergillus, Pichia, Hansenulaor Yarrowia, even more preferably selected from Escherichia coli,Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus ruber,Rhodococcus equi, Pseudomonas putida, Saccharomyces cerevisiae,Aspergillus niger, Pichia pastoris, Hansenula polymorpha or Yarrowialipolytica, most preferably selected from Escherichia coli B, inparticular E. coli BL21 (DE3) or other derivatives or E. coli K-12.

(13) A process for the production of vitamin E, comprising the step ofbiocatalytic conversion of α-isophorone into KIP according to any ofembodiments (2), (3) or (4).

(14) A process according to embodiment (13), wherein the biocatalyticconversion of α-isophorone into KIP is performed as one-pot two-stepprocess.

The present invention is now described in greater detail with referenceto FIGS. 1 to 9 and the following examples. The work leading to thisinvention has received funding from the European Union (EU) projectROBOX (grant agreement no 635734) under EU's Horizon 2020 ProgrammeResearch and Innovation actions H2020-LEIT BIO-2014-1.

FIG. 1: FIG. 1A shows the proposed one-pot double allylic oxidation,wherein α-isophorone (α-IP, 1) is in a first step oxidized via enzymaticaction of a cytochrome P450 enzyme (P450) leading to4-hydroxy-α-isophorone (HIP, 6), which might be further oxidized viaenzymatic action of aldehyde dehydrogenase (ADH) to2,6,6-trimethylcyclohex-2-ene-1,4-dione (KIP, 3). For furtherexplanation see in the text. Possible products of the P450 oxidation ofα-IP (1) are HIP (6), HIMP (7) and IPO (8) shown in FIG. 1B. Theregeneration of CM-ADH10 catalyzing the oxidation from (R)-HIP to KIPusing different co-substrates such as acetone (9a), chloroacetone (9b),ethyl acetoacetate (9c) and ethyl levulinate (9d) leading to therespective reduction products 10a-d, wherein “R” defines the respectivesubstituent to result in the different co-substrates 9a-d is shown inFIG. 1C. Whole-cell double oxidation cascade of α-IP to KIP using E.coli co-expressing P450-WAL and Cm-ADH10 is shown in FIG. 1D. For moreexplanation, see text.

FIG. 2: Comparison of TTN as shown of the x-axis for HIP (black bars)and HMIP (grey bars) obtained for P450cam-RhFRed pooled variants shownon the y-axis. For further explanation see in the text.

FIG. 3: Comparison of TTN as shown of the x-axis for HIP (black bars)and HMIP (grey bars) obtained for library D (mutated locations) shown onthe y-axis. The data is based on the wild-type P450cam-RhFRed carryingthe substitution Y96F. The introduction of F87W in said backgroundtogether with L244A-V247L led to more than a 6-fold improvement of theTTN values with respect to L244A-V247L mutant. For further explanationsee in the text.

FIG. 4: Bar chart showing conversion values obtained with different HIPconcentration. Reaction set-up: 1 mg/mL purified Cm-ADH10, 0.25 mM NADP+and 0.5% v/v ethyl acetoacetate as co-substrate for cofactorregeneration (30° C., 24 h). Product concentrations (mM) are given aboveeach bar. Conversion of HIP in % is shown on the y-axis, HIPconcentration in mM is shown on the x-axis. For further explanation seein the text.

FIG. 5A: Effect of buffer concentration and pH on Cm-ADH10 activity.Reaction set-up: 1 mg/mL purified Cm-ADH10, 0.25 mM NADP+ and 0.5% v/vethyl acetoacetate as co-substrate for cofactor regeneration (30° C.,4.5 h). For further explanation see in the text.

FIG. 5B: Effect of the pH reached after the P450-catalyzed reaction onCm-ADH10 performance. KPi buffer concentration used in the first step isgiven on the x-axis (“1”=50+100 mM KC; “2”=100 mM; “3”=200 mM; “4”=300mM). Black bars: HIP conversion after direct addition of Cm-ADH10 (thepH measured after the first step is indicated above). Grey bars: HIPconversion when the supernatant of the first reaction was titrated to pH8.0 before Cm-ADH10 addition. For more explanation, see text.

FIG. 6: Optimization of the allylic oxidation of α-IP catalyzed byP450cam-RhFRed-WAL. FIG. 6A shows the effect of buffer concentration.Reaction set-up: 200 mg mL⁻¹ wet cells resuspended in KPi buffer pH 8.0(concentration given on the x-axis), 10 mg mL⁻¹ glucose, 20 mM α-IP, 2%DMSO, 20° C., 1 mL final volume in deep-well plate, 24 h reaction time.FIG. 6B shows effect of temperature. Reaction set-up: same as above,exception being the buffer adopted (200 mM KP, pH 8.0) and substrateconcentration (15 mM) The x-axis shows the temperature (up to 40° C.),the y-axis shows α-IP conversion in %.

FIG. 7: Optimization of the oxidation of (R)-HIP catalyzed by Cm-ADH10.The y-axis shows HIP conversion in %. FIG. 7A shows the effect ofdifferent co-substrates. Reaction set up: 100 mM KPi buffer pH 7.5, 1 mgmL⁻¹ Cm-ADH10, 0.25 mM NADP+, 0.5% v/v co-substrate, 40 mM (R)-HIP, 30°C. FIG. 7B shows the effect of pH as indicated on the x-axis. Reactionset up: 100 mM indicated buffer, 1 mg mL⁻¹ Cm-ADH10, 0.25 mM NADP+, 0.5%v/v chloroacetone, 40 mM (R)-HIP, 30° C., 4 h. FIG. 7C shows the effectof temperature as indicated in ° C. on the x-axis. Reaction set up: 200mM KPi buffer pH 8.0, 1 mg mL⁻¹ Cm-ADH10, 0.25 mM NADP+, 0.5% v/vco-substrate, 40 mM (R)-HIP, 24 h. For further details see text or FIG.1C.

FIG. 8: Effect of buffer concentration and pH on Cm-ADH10 activity.Reaction set-up: 1 mg/mL purified Cm-ADH10, 0.25 mM NADP+ and 0.5% v/vethyl acetoacetate as co-substrate for cofactor regeneration (30° C.,4.5 h). HIP-conversion in % is shown on the y-axis, the concentration ofKPi buffer in mM is shown on the x-axis.

FIG. 9: Time-course experiment for the one-pot two-step double allylicoxidation of α-IP. Reaction set up: 200 mg mL⁻¹ wet cells resuspended in200 mM KPi buffer pH 8.0, 10 mg mL⁻¹ glucose, 10 mM α-IP, 2% DMSO (firststep, 28° C.), then addition of 20% v/v Cm-ADH10 cell-free extract and1.6 eq. of chloroacetone to the supernatant of the first reaction(second step, 40° C.). The arrow indicates the time of addition.Composition in % is shown on the y-axis, Times per h is shown on thex-axis.

EXAMPLE 1: GENERAL METHODOLOGY

All chemicals solvents, and carbon monoxide for CO differencespectroscopy used were of analytical grade and purchased from SigmaAldrich (Poole, Dorset, UK) or BOC gases (Guildford, UK). Competentcells and enzymes were received from New England Biolabs (NEB). M9minimal salts (5×) were purchased from Sigma-Aldrich, reconstituted bystirring the recommended amount of powder in water and sterilized byautoclaving. 40% glucose (w/v), antibiotics 1000×, 1 M MgSO₄, 1 MCaCl₂), and 25% (w/v) FeCL were prepared in dH₂O and filter sterilizedthrough a 0.2 μm syringe filter.

Inverse PCR reactions carried out using Eppendorf Mastercycler Gradientthermal cyclers according to NEB guidelines, followed by DpnI digestionbefore carrying out ligation reaction for 1 h at 25° C. with T4 DNAligase and polynucleotide kinase, according to the manufacturersinstruction. NEB 5-alpha competent E. coli (high efficiency) were thentransformed according to the manufacturer instruction and sequenceverified by plasmid sequencing. Expression plasmids were generated bystandard restriction cloning. P450cam-RhFRed site-directed mutants weremade starting from variants in the previously developed libraries (seeExample 2) using the appropriate primers shown in Table 1. Primersynthesis and DNA sequencing were performed by Eurofins Genomics. AnAvril restriction site was added to P450cam-RhFRed by PCR using theprimers “17 for modified” and “AvrII rev” and the sequence cloned inpCDF-1b using NcoI and Avril restriction sites. For the two-vectorstrategy for coexpression (or ADHs expression trials), 55CR and Cm-ADH10were cloned into pET28a vector, using NdeI and XhoI restriction sitesthat were introduced by PR using primers “NdeI Sporo for” and “XhoISporo rev” or “NdeI ADH10” for and “XhoI ADH10 rev”, respectively.Plasmid carrying genes encoding for the selected ADHs were kindlyprovided by c-LEcta GmbH, Leipzig, Germany.

TABLE 1. Oligonucleotides used for inverse PCR reactions. Mismatching bases are underlined. Primer SEQ sequence IDPrimer 5′-3′ NO: F87W for, GAGTGCCCGTGGA 9 Tm = 57° C. TCCCTCGTGaaGCF87W rev, GCTGGAAAAGT 10 Tm = 57° C. GGCGGTAATC L244A for,AGGATGTGTGGCGC 11 Tm = 59° C. GTTACTGGTCGGC L244A rev, CTTGGCTTCGTCA 12Tm = 61° C. CTGGTGATCG L244A-V247L for, AGGATGTGTGGCGCG 13 Tm = 63° C.TTACTGCTCGGCGGC CTGGATAC AvrII rev, TAGTCTCCTAGGTCAG 14 Tm = 65° C.AGTCGCAGGGCCAGCc AflII rev, TCGTCTCTTAAGTCAG 15 Tm = 65° C.AGTCGCAGGGCCAGCC T7 for modified, TAATACGACTCACTAT 16 Tm = 62° C.AGGGAGACCACAACGG NdeI ADH10 for, ACGTAGCATATGACGA 17 Tm = 63° C.CTACTTCAAACGCGCT TGTC NcoI CmADH for, ATGCTACCATGGGGAT 18 Tm = 63° C.GACGACTACTTCAAAC GCGCTTGTC AflII CmADH rev, ATGTATCTTAAGCAAT 19Tm = 60° C. CAAGCCATTGTCGACC AC XhoI ADH10 rev, ACGTCACTCGAGTTAA 20Tm = 60° C. GCAATCAAGCCATTGT CGACCAC AvrII ADH10 rev, AGTCAGCCTAGGTTAA21 Tm = 60° C. GCAATCAAGCCATTGT CGACCAC NdeI Sporo for,CTGGATCATATGGCCAA 22 Tm = 60° C. AATCGATAATGCCGTG XhoI Sporo rev,TGCATGCTCGAGTTAG 23 Tm = 61° C. GCTGTTTCGCTACCAA CCAGG

Chiral normal HPLC for measurement of enzymatic activity was carried outon an Agilent System (Santa Clara, Calif., USA) equipped with a G4225Adegasser, G1311A quaternary pump, a G1329A well plate autosampler unit,a G1315B diode array detector and a G1316A thermostatted columncompartment. Separation of (S)- and (R)-HIP was carried out using aCHIRALPAK® AS-H column (5 μm particle size, 4.6 mm diameter×250 mm;Daicel Chemical Industries Ltd) operating in isocratic mode with 80%hexane and 20% isopropanol for 18 min at 25C. Injection volume was 5NLand chromatograms were monitored at 254 nm. Retention times were asfollow: KIP 9.5 min, (S)-HIP 10.9 min α-IP 12.4, (R)-HIP 13.3 min.Automated GC analysis was performed on an Agilent 6850 GC (Agilent,Santa Clara, Calif., USA) with aflame ionization detector (FID) equippedwith an Agilent HP-1 column (30 m length, 0.32 mm inner diameter, 0.25μm film thickness, Agilent, Santa Clara, Calif., USA). 2 μL of propertydiluted sample was injected at a split ratio 10:1. The inlet temperaturewas set at 200° C., detector temperature at 250° C. and pressuremaintained at 6.8 psi. The following method was applied: initialtemperature 50° C.; 10° C./min to 220, hold 2 min. The correspondingretention times were: α-IP 7.4 min, KIP 7.6, possible KIP-reductionby-product 7.8 min and HIP 9.7 min. Calibration curves of decane vs 6 or3 were constructed in order to calculate TTNs or conversion values,respectively.

Protein activity measurement of the P450 BM3 mutant libraries isdescribed in more details in Examples 1 of WO2013160365. The wild-typeP450 BM3 is shown in SEQ ID NO:3, with the encoding nucleic acidsequence shown in SEQ ID NO:4.

Whole cell P450 concentration measurement was performed on a platereader (Tecan Infinite 200 series, Männedorf, CH) according to Kelly etal. (Beilstein J. Org. Chem. 2015, 11, 1713-1720) and for cell lysateson a Cary 50 UV/Visible spectrophotometer (Agilent Technologies, SantaClara, Calif., USA) according to the protocol by Omura and Sato (J.Biol. Chem. 1964, 239, 2370-2378). NMR spectra were recorded on a BrukerAvance 400 spectrometer (400.1 MHz for 1H and 100.6 MHz for 13C) indeuterated chloroform.

Preparation of (R)-HIP and (S)-HIP were prepared according to Hennig et.al. (Tetrahedron: Asymmetry 2000, 11, 1849-1858). Benzeneruthenium(II)chloride dimer (10.5 mg) and (1S,2R)-2-Amino-1,2-diphenylethanol (17.7mg) were dissolved in 5 mL of isopropanol and the red solution stirredfor 30 min at 80° C. Afterwards, 36.5 mL of isopropanol were added andthe solution cooled to 28° C. Then, ketoisophorone (631.6 μL) and NaOH(2 mL, 0.1 M in isopropanol) were added to give a darker solution andthe reaction followed until completion (ca. 3 hours). The reactionmixture was then filtered through Celite and the filtrate evaporated toafford a black oily residue. The product (S)-HIP was then purified bysilica gel chromatography. The column was successively eluted withcyclohexane containing 20, 50 and 70% ethyl acetate and then solventsevaporated to afford a dark green oily residue (253.4 mg, 40% isolatedyield). For the preparation of (R)-HIP, the same procedure was followed,but (1R,2)-2-Amino-1,2-diphenylethanol was used.

Expression and purification of proteins was as follows: plasmids(pET-14b, pCDF-1b or pET-28a) carrying genes encoding P450cam-RhFRedvariants were stored at 4° C. Chemically competent E. coli BL21 (DE3)cells were transformed by heat shock according to manufacturerinstructions. Transformants were plated on LB agar with added antibiotic(150 μg/ml ampicillin or 50 μg/ml for spectinomycin and kanamycin) andgrown at 37° C. for 16 hours. Single colonies were picked from plates toprepare starter cultures in LB medium supplemented with antibiotic.After 16 hours, expression cultures were inoculated 1/100 using LBstarter cultures and, to guarantee proper aeration, cultures volume wasno more than 25% of the total conical flask volume. P450 expression inminimal medium was carried out according to Kelly et al. (supra): 1×M9salts solution were supplemented with 0.4% glucose, 0.05% of FeCl₃, 1 mMMgSO₄, 1 mM CaCl₂) and cells were grown with vigorous shaking (200 rpm)at 37° C. until an OD600 of 0.8 was reached. At this stage,isopropyl-D-1-thiogalactopyranoside (IPTG, 0.4 mM) was added to induceprotein expression, along with ALA (0.5 mM) supplementation. Proteinexpression was carried out at 20° C. with shaking at 200 rpm for 20 h.The use of complex media (e.g., LB, TB and auto-induction media)resulted in the expression of the protein in the insoluble fraction orsoluble inactive protein (data not shown).

Similarly, production of Cm-ADH10 and 55CR was carried out following aprotocol similar to that for P450cam-RhFRed expression, the exceptionbeing the medium used (TB instead of M9) and the need for 5-ALAsupplementation (not added). SDS-polyacrylamide gel electrophoresis(SDS-PAGE) was employed to confirm protein expression. RecombinantCm-ADH10 was obtained by inoculating 1 L of Terrific broth containing 50μg/mL kanamycin with transformed cell E. coli BL21 (DE3) and incubateduntil an OD600 of 0.6; protein production was induced with 0.4 mM IPTGat 20° C. and production was kept for 16 h. Cells were harvested bycentrifugation (2831 g, 20 min, 4° C.) and kept at −80° C. untilpurification. Cells from 1 L of culture were suspended in 50 mL of 20 mMTris-HCl, pH 7.5. The crude extract was prepared by sonication 5 min,70% amplitude, 5 s on/off. Cell debris was removed byultracentrifugation at 31,000 g for 45 min, 4° C. The supernatant wasfiltrated using a 0.45 um filter. The cell-free extract was applied to a5 mL HisTrap FF column using an AKTA Pure (GE healthcare) at 1 ml/min at4° C. and a 20 mM imidazole solution in 20 mM Tris-HCl buffer, pH 7.5.The column was washed with 4-6 CV of a 50 mM imidazole solution in 20 mMTris-HC buffer, pH 7.5. Pure enzyme was eluted using 3 CV of a 500 mMimidazole solution in 20 mM Tris-HC buffer, pH 7.5. Enzyme wasconcentrated and excess of imidazole was removed with a concentratorAmicon® Ultra concentrator (10,000 NMWL; Millipore) at 4000 g.Typically, the enzyme was diluted to 10 mg/mL in 50 mM KPi buffer (pH7.5) to carry out biotransformation trials and kinetic measurements,whereas for crystallographic studies, concentrated aliquots (1-1.5 mL)were treated with thrombin (0.5 U, 4° C., overnight shacking) andcleaved 6×-His Tag was removed by gel filtration in a Superdex 200 10/30GL. Pure protein, as judged by UV chromatogram and SDS-PAGE wasconcentrated to 77 mg/mL and stored at −80° C.

For screening and characterization of ADHs, a panel of alcoholdehydrogenases enzymes as freeze-dried cell free extracts in 96-deepwell plates were kindly supplied by c-LEcta GmbH (Leipzig, Germany). Fora first screening, each freeze-dried extract was resuspended in 50 μL of50 mM sodium phosphate buffer pH=7.2, 100 mM KC, and reaction carriedout on a 500 μL scale with both 10 mM (R)- and (S)-HIP, 0.5 mM of bothNAD+ and NADP+ and acetone 5% v/v (30° C.).

Then, best hits were chosen for a further screen on a 500 μL scale with10 mM (R)-HIP, 0.5 mM NADP+ and acetone 5% v/v. Reaction mixtures wereextracted with methyl-tert-butyl ether (MTBE), vortexed for 30 s andcentrifuged 5 minutes before removing the organic layer, which was thentransferred to fresh tubes containing MgSO₄. Finally, 250 μL of the MTBEextract was taken for chiral normal phase HPLC analysis.

For protein crystallization and docking experiments, native Cm-ADH10 wascrystallized at 293 K using the sitting-drop vapour diffusion techniqueat 20° C. Equal volumes of 12 mg/mL Cm-ADH10 in 20 mM Tris-HCl at pH 7.5and reservoir solution were mixed. The reservoir solution contained 40%polyethylene glycol (PEG) 200 in 0.1 M MES at pH 6.5 (w/v). Initialconditions were screened using the JSCG, PEG and ammonium sulphatescreens (Qiagen) in 96-well sitting drop trays. X-Ray diffraction datawere collected at the ID23-2 beamline of the European SynchrotronRadiation Facility in Grenoble, France (ESRF). The images wereintegrated and scaled using MOSFLM (Battye et al., Acta Crystallogr.Sect. D Biol. Crystallogr. 2011, 67, 271-281). Intensities were mergedand converted to amplitudes with Aimless (Evans and Murshudov, ActaCrystallogr. Sect. D Biol. Crystallogr. 2013, 69, 1204-1214) and othersoftware of the CCP4 Suite (Winn et al., Acta Crystallogr. Sect. D Biol.Crystallogr. 2011, 67, 235-242). The structure was solved with MOLREP(Vagin and Teplyakov, J. Appl. Cryst 1997, 30, 1022-1025) and thecoordinates of the dehydrogenase/reductase from Sinorhizobium meliloti1021 (PDB: 3V2G) as the search model. AutoDock Vina (Trott and Olson,supra) was used to run ligand-receptor docking calculation of (R)-HIP onCm-ADH10. Structures were prepared with DockPrep (Lang et al., Rna 2009,1219-1230) in Chimera. The best model yielded a score of −5.6 kcal/mol,a rmsd (l.b) of 0.0 and a rmsd (u.b.) of 0.0.

Example 2: Screening and Improvement of P450cam-RhFRed Variants

A plasmid library encoding 96 P450cam-RhFRed mutants generated in aprevious work (Kelly et al., Beilstein J. Org. Chem. 2015, 11,1713-1720) were purified from bacterial glycerol stocks. P450cam-RhFRedvariants were produced targeting 7 pairs of residues by saturationmutagenesis using NDT codon degeneracy. This resulted in the productionof seven libraries: A to F (see Table 2). In order to cluster thesevariants, 100 ng of each plasmid purified were added to one of fivepools according to the mutations introduced, the exceptions beingvariants from library E, F and G that were pooled together because thereduced size of these libraries (giving five pooled libraries in total).These plasmids preparations were then transformed into E. coliNEB-5-alpha competent cells and plasmids purified again fortransformation into E. coli BL21 (DE3) for expression. FIG. 2 shows themeasured biocatalytic performance expressed in terms of the total TTN,that is the ratio between total product concentration (in μM) andcatalyst concentration (in μM), calculated over 24 h. Finally, theenantiomeric excess values (ee) were also determined by comparison withsynthesized standards (see Table 2). The P450-catalysed single oxidationof α-IP may lead to (at least) three major products (FIG. 1B): HIP (6),the regioisomeric allyic oxidation product(3-(hydroxymethyl)-5,5-dimethylcyclohex-2-en-1-one, HMIP, 7) andisophorone oxide (2,3-epoxy-3,5,5-trimethyl-1-cyclohexanone, IPO, 8).Product 7 was identified after scale up and NMR analysis (not shown).The formation of isophorone oxide was not observed and (R)-HIP was thepreferred product (FIG. 1B), with library D (mutations at residuesL244-V247) showing the highest TTNs in the desired α-IP allyticoxidation. Notably, no activity was detected for mutants in pool C(mutations at residues M184-T185).

TABLE 2 Possible products of the P450 oxidation of α-IP and comparisonof (R)-HIP ee values for the different pooled mutation libraries, “nd”means “not detectable”. Pool Position of mutation (R) HIP ee value EFGG248-T252; V295-D297; I395-V396 77% D L244-V247 94% C M184-T185 n.d. BF98-T101 48% A F87-F96 >99% 

These initial results drove the next step of the investigation towardsthe single-clone level analysis of mutants at positions L244-V247.

Expression cultures were centrifuged (2831 g, 20 min, 4° C.) and thepellet resuspended to 230 mg/mL (wet weight) in the appropriatebiotransformation buffer (50 mM sodium phosphate buffer, pH=7.2, 100 mMKCl).

Biotransformations were carried out in 48 well plates on a 1 ml scale,with 880 μL of re-suspended cells, 100 μL of 100 mg/mL glucose and theappropriate final concentration of substrate typically added fromconcentrated stocks in DMSO (2% v/v final concentration). Plates weresealed with a gas permeable membrane and reaction carried out with 250rpm orbital shaking for 24 h. Reaction mixtures were extracted with 1 mLof methyl-tert-butyl ether (MTBE) with 1 mM decane (as internalstandard) vortexed for 30 s and centrifuged 5 minutes before removingthe organic layer, which was then transferred to fresh tubes containingMgSO₄. Finally, 250 μL of the MTBE extract was taken for GC analysis anddiluted when necessary. In order to determine the ee %, the samples werealso analyzed by chiral normal phase HPLC (see Ex. 1).

Analysis of biotransformation products (FIG. 3) demonstrated thatpositions 244 and 247 greatly affect TTNs of α-IP. Interestingly, themajority of mutants bearing the bulky aromatic phenylalanine residueshow not only reduced TTNs but also a decrease in the regioselectivityof the reaction, with the formation of HIP and HMIP in approximatelyequal amounts by mutants L244Y-V247F and L244I-V247F. Given the observedpositive effect of the substitution of L244 with small or apolarresidues, the mutation L244A was also introduced, considering the effectof L244A mutation on substrate acceptance, regio- andenantioselectivity. The primers for said mutagenesis reactions arelisted in Table 1. The protocol is described in Example 1. The proteinand DNA sequences of P450cam-RhFRed wild-type enzyme carrying the aminoacid substitution Y96F are shown in SEQ ID NO:1 and 2, respectively.

This mutation was studied either in presence of the wild-type V247 orV247L, leading to good TTNs values. The two mutants L244A-V247 andL244A-V247L showed the best TTN values (94±9 and 83±11, respectively)and were selected for further engineering. Furthermore, the bulkytryptophan was introduced in place of phenylalanine at position 87. Asshown in Table 3, the introduction of F87W in the L244A-V247L backgroundled to more than a 6-fold improvement of TTNs values with respect toL244A-V247L mutant, with no HMIP detected and an ee of 99% (R)-HIP,suggesting a better orienting effect of this biocatalyst towards thehighly reactive iron-oxo species. Finally, the best improved variant(Y96F-F87W-L244A-V247L, now termed P450-WAL) was selected for subsequentoptimization experiments for the designed cascade reaction (seeExample 1) The results are shown in Table 3.

TABLE 3 (R)-HIP ee values obtained for different mutations from libraryD. Note that the strain already carries the Y96F mutation. Mutant (R)HIP ee value L244N-V247 94% L244S-V247L 99% L244N-V247L 99% L244G-V247L99% L244I-V247L 98% L244C-V247L 98% L244I-V247F 95% L244Y-V247F 93%L244N-V247F 98% L244H-V247F 99% L244G-V247F 96% L244I-V247I 98%L244I-V247S 59% L244A-V247L 97% L244A-V247 84% F87W-L244A-V247 82%F87-L244A-V247L 99%

Example 3: Screening and Characterization of HIP-Oxidizing ADHs

A screening kit of 116 ADHs from very diverse organisms and with a broadrange of accepted substrates was provided by c-LEcta. In order toidentify suitable biocatalysts for the oxidation of (R)-HIP, the panelwas screened against this substrate by HPLC (see Example 1). Severalenzymes (>80%) showed almost no activity, due to the steric hindrance ofthe substrate, however, among the positive hits, two were selected forhaving the highest activity: Cm-ADH10 from Candida magnoliae (GeneBankaccession no. AGA42262.1; SEQ ID NO:5) and the NADPH-dependent carbonylreductase (55CR) from Sporobolomyces salmonicolor (UniProt accession no.Q9UUN9; SEQ ID NO:7). Notably, both enzymes accept NADP(H) as cofactor,which is desirable to create a self-sufficient cascade with respect tothe cofactor. The corresponding genes were cloned into a pET28a vectorto carry out expression trials (see Example 1), which revealed goodexpression levels for Cm-ADH10. Unfortunately, expression of 55CR wasvery low (not shown). The respective nucleotide acid sequences are shownin SEQ ID NO:6 and 8, respectively.

To understand better the catalytic properties of Cm-ADH10, we solved thecrystal structure of its complex with NADP+ to a resolution of 1.6 Å(see Example 1; data not shown). After soaking the crystal with asolution of (R)-HIP (10 mM), no substrate was bound to the crystal.However, after docking (AutoDock Vina), the most favored calculation forthe bound ligand (R)-HIP confirms that the cavity can accommodate thesubstrate at a distance and geometry that would favor the transfer of ahydride from the chiral carbon on the 4R-hydroxy moiety to C4N of NADP+.In this position, the 4-hydroxy group would be in the right geometry tointeract with the catalytically important residues 5144 and Y157, whilethe rest of the molecule of 4R-HIP is possibly stabilized byinteractions with residues H149 and Y189 in the entrance of the activesite. Coincidentally, this area in the crystal structure is occupied bya tetrad of water molecules that have been reported to be bound to theTyr-OH and Lys side chain, thus mimicking substrate and ribose hydroxylgroup positions.

Preliminary biotransformations by varying (R)-HIP concentration from 10to 100 mM were carried out, using 1 mg mL⁻¹ purified Cm-ADH10 with aconstant NADP+ concentration (0.25 mM) and 0.5% v/v ethyl acetoacetateas co-substrate for cofactor regeneration by the same ADH acting as adual-functional enzyme. With this setup, conversions ranged from 92%with 10 mM substrate, down to 48% conversion with 100 mM substrate (seeFIG. 4). Thus, Cm-ADH10 lends itself to being applied in the designedbi-enzymatic cascade.

Example 4: Optimization of Buffer Concentration, pH, and Temperature

Initially, we tried to combine the two selected biocatalysts in aone-pot, two-step format, adding 1 mg mL⁻¹ of purified Cm-ADH10,cofactor and co-substrate (see above) directly to the supernatant of thewhole-cell P450 reaction carried out with 200 mg mL⁻¹ wet cell load in50 mM sodium phosphate buffer pH 7.2, 100 mM KC (indicated as standardbiotransformation buffer) and 10 mg mL⁻¹ glucose for cofactorregeneration by E. coli metabolism. Unexpectedly, we obtained very lowconversion values for the ADH catalyzed reaction, starting from just 10mM α-IP in the first step. We reasoned that potential inhibitors for thesecond step could originate from the growth of the whole-cellbiocatalysts in the M9 minimal medium supplemented with glucose (seeFIG. 5A). The pH of the supernatant resulting from the first oxidationstep carried out in phosphate buffer with different concentration wasmeasured (FIG. 5B). Surprisingly, the pH dropped by 1.5 units when thestandard biotransformation buffer was used, and even with a 300 mMpotassium phosphate (KPi) buffer the pH decrease was significant.Nevertheless, by increasing the capacity of the buffer employed in thefirst step, higher conversions where observed in the second one.Eventually, more than a 2-fold increase in HIP conversion was observedwhen P450 catalyzed reaction carried out in standard biotransformationbuffer were titrated to pH 8.0 before the addition of Cm-ADH10. Asimilar trend was observed when cells grown in M9 medium where simplyresuspended in buffer without any addition of substrate or glucose,suggesting that the observed pH decrease is linked to the metabolism ofcells grown to high density (OD600—5.0-6.5). Thus, in order to establisha one-pot two-step process there is a need for a careful optimization ofthe each individual step before combination.

Next, we proceeded with the parallel optimization of the two oxidativesteps with respect to buffer concentration, pH and temperature. For theP450 allylic oxidation step, KPi buffer (pH 8.0) was chosen for theunique K+ binding site of P450cam, which displays higher stability andsuperior camphor binding in presence of K+ ions. The effect of bufferconcentration was examined, along with the temperature optimum. As shownin FIG. 6A, conversion improved with increasing buffer concentration upto 200-300 mM, and by comparing biotransformations carried out in 50 mMKPi without KC, with KCL or in 100 mM KPi, it can be concluded thatbuffer capacity seems to have a greater effect on conversion than K+concentration. Eventually, 200 mM KPi buffer was chosen to investigatethe effect of temperature on conversion, finding the optimum at 28° C.(FIG. 6B).

With respect to the Cm-ADH10 alcohol oxidation, different co-substrateswere selected and tested in order to exploit the biocatalyst as adual-functional enzyme capable of performing the whole substrateoxidation-cofactor regeneration cycle (FIG. 7A). Besides testing acetone(9a), we have also included activated ketones such as chloroacetone(9b), ethyl acetoacetate (9c) and ethyl levulinate (9d), since theirreduction (and hence cofactor regeneration) is favored by an additionalintramolecular hydrogen bond between the newly formed alcohol functionalgroup and the electronegative moiety.

Chloroacetone and ethyl acetoacetate proved to be superior and, even ifthe latter performed better during the first six hours of reaction, theformer pushed the conversion to 84% with 40 mM substrate after 24 h (vs.67% with ethyl acetoacetate). Therefore, chloroacetone was employed ashydrogen acceptor in all the subsequent optimization steps. The pHeffect on the reaction outcome (FIG. 7B) partially explains our initialunsuccessful attempts to combine the two enzymes: in fact, enzymeactivity drops below pH 6.0, with an optimum pH range between 7.0 and9.0. Moreover, the buffer system employed in the P450 catalyzed reaction(200 mM KPi) displayed perfect compatibility with the second step (FIG.8). Finally, we turned our attention to temperature optimization,finding the highest conversion at 40° C. (FIG. 7C).

With these optimized conditions in hand, we moved on to the envisagedbi-enzymatic cascade concept to carry out scale-up experiments forproduct isolation and characterization.

Example 5: Double Oxidation from α-Isophorone to Ketoisophorone

In order to further simplify the entire process, the oxidation of(R)-HIP was accomplished using a concentrated cell-free extract (CFE) ofE. coli overexpressing Cm-ADH10, thus avoiding the expensive proteinpurification step. The course of the reaction under the optimizedconditions was followed over a total reaction time of 50 h, showing thatthe P450 catalyzed allylic oxidation of 10 mM α-IP reached 94±2%conversion in 18 h, whereas the second step was much faster, reachingcomplete conversion over 6 h (FIG. 9). Remarkably, the addition of NADP+for the second step was found to be unnecessary to achieve the highestconversion, further reducing process costs. Some degree of overoxidationof HIP to KIP was observed during the first step.

Given these results, we have also attempted to co-express theP450cam-RhFRed-WAL variant with Cm-ADH10 in the same host with atwo-vector system to carry out the whole-cell double oxidation of α-IP(FIG. 1D). After 24 h, the conversion reached an average value of 732%,approximately 20% lower than for the one-pot two-step process. However,the co-expression of the two enzymes and cofactor preferences make thisreaction redox-balanced, avoiding addition of a CFE and co-substrate. Infact, P450 expression level was reduced 3-fold in the “designer”microorganism (6.0 μM for the two-step process vs. 1.9 μM for thewhole-cell process), which accounted for the residual α-IP (not shown).

1. A one-pot biocatalytic process for the conversion of α-isophoroneinto ketoisophorone (KIP) comprising the enzymatic steps of: (a)conversion of α-isophorone into 4-hydroxy-α-isophorone (HIP) with aconversion rate of at least 80% via catalytic action of a P450monooxygenase selected from a polypeptide having at least 35% identityto SEQ ID NO:1 or a polypeptide having at least 62% identity to SEQ IDNO:3, said P450 monooxygenase comprising one or more mutation(s) on aposition corresponding to position(s) 96, 87, 244, 247, and/orcombinations thereof in P450cam-RhFRed P450 monooxygenase according toSEQ ID NO:3, and wherein the total turnover number is increased by atleast 2-fold compared to the respective non-modified P450 monooxygenaseunder incubation conditions of pH of from 4.0 to 10.0 for 1 to 48 h withoptionally isolation of HIP from the reaction mixture; and (b)conversion of HIP into KIP via catalytic action of an enzyme havingalcohol dehydrogenase or carbonyl reductase activity, preferably analcohol dehydrogenase from Candida magnoliae or a carbonyl reductasefrom Sporobolomyces salmonicolor, said suitable conditions includeincubation at pH of from 4.0 to 10.0 for 1 to 48 h with optionallyisolation of KIP from the reaction mixture.
 2. A process according toclaim 1, wherein the biocatalytic process for the conversion ofα-isophorone to 4-hydroxy-α-isophorone (HIP) and/or the conversion ofHIP to ketoisophorone (KIP) is carried out in the presence of (a) aco-substrate selected from the group consisting of glucose, isopropanoland phosphite with regards to conversion of α-isophorone to HIP; or (b)a co-substrate selected from the group consisting of acetone,chloroacetone, ethyl acetoacetate, ethyl levulinate, chloroacetone andethyl acetoacetate with regards to conversion of HIP to KIP.
 3. Aprocess according to claim 1, wherein the enantiomeric excess towardsthe (R)-configuration of HIP and/or KIP generated via the biocatalyticprocess is at least 50% based on the total amount of HIP and/or KIP. 4.A process according to claim 1 which is performed at a temperature about30° C. to 40° C.
 5. A modified P450 monooxygenase for use in a processaccording to claim 1 comprising one or more mutation(s) on a positioncorresponding to position(s) 96, 87, 244, 247, and/or combinationsthereof in P450cam-RhFRed P450 monooxygenase according to SEQ ID NO:3,wherein: (a) the introduced amino acid on a position corresponding toposition 244 are selected from the group consisting of alanine,asparagine, serine, glycine, isoleucine, cysteine, tyrosine andhistidine; and/or (b) the introduced amino acid on a positioncorresponding to position 247 are selected from the group consisting oflysine, phenylalanine, isoleucine and serine; and/or (c) the introducedamino acid on a position corresponding to position 87 is tryptophan;and/or (d) the introduced amino acid on a position corresponding toposition 97 is phenylalanine.
 6. A modified P450 monooxygenase accordingto claim 5, wherein: (a) the introduced amino acid on a positioncorresponding to position 244 is selected from alanine and/or (b) theintroduced amino acid on a position corresponding to position 247 isselected from lysine; and/or (c) the introduced amino acid on a positioncorresponding to position 87 is tryptophan; and/or (d) the introducedamino acid on a position corresponding to position 97 is phenylalanine.7. A modified P450 monooxygenase according to claim 5, comprisingcombinations of amino acid substitutions on a position corresponding toposition(s) 244 and 247 in P450cam-RhFRed P450 monooxygenase accordingto SEQ ID NO:3 which are selected from L244A-V247 and L244A-V247L.
 8. Apolynucleotide sequence comprising a DNA sequence coding for a P450monooxygenase according to claim
 5. 9. A host cell wherein a P450monooxygenase according to claim 5 is expressed, said host beingselected from the group consisting of bacteria, fungi, yeasts or plantor animal cells, more preferably selected from Escherichia,Streptomyces, Bacillus, Rhodococcus, Pseudomonas, Saccharomyces,Aspergillus, Pichia, Hansenula or Yarrowia, even more preferablyselected from Escherichia coli, Rhodococcus erythropolis, Rhodococcusrhodochrous, Rhodococcus ruber, Rhodococcus equi, Pseudomonas putida,Saccharomyces cerevisiae, Aspergillus niger, Pichia pastoris, Hansenulapolymorpha or Yarrowia lipolytica, most preferably selected fromEscherichia coli B, in particular E. coli BL21 (DE3) or otherderivatives or E. coli K-12.
 10. A process for the production of vitaminE, comprising the step of biocatalytic conversion of α-isophorone intoKIP according to claim 1.